Variability in commercial ginseng products: an analysis of 25 preparations 1 3

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1 Variability in commercial ginseng products: an analysis of 25 preparations 1 3 Martha R Harkey, Gary L Henderson, M Eric Gershwin, Judith S Stern, and Robert M Hackman ABSTRACT Background: Because dietary supplements are not subject to the same regulations that pharmaceuticals are, there is concern among medical professionals that these products may lack purity or potency. Objective: To determine the variability in a range of ginseng herbal products available in the United States, we identified and measured the concentration of marker compounds by using HPLC and liquid chromatography tandem mass spectrometry. Design: Twenty-five commercial ginseng preparations from the genera Panax or Eleutherococcus were obtained from a local health food store and analyzed for 7 ginsenosides (marker compounds for Panax species, which include Asian and American ginseng) and 2 eleutherosides (marker compounds for Eleutherococcus senticosus, also known as Siberian ginseng). Results: All plant products were correctly identified by botanical plant species (ie, Panax species or E. senticosus); however, concentrations of marker compounds differed significantly from labeled amounts. There was also significant product-to-product variability: concentrations of ginsenosides varied by 15- and 36-fold in capsules and liquids, respectively, and concentrations of eleutherosides varied by 43- and 200-fold in capsules and liquids, respectively. Although a systematic search for adulterants was not conducted, review of the HPLC and liquid chromatography tandem mass spectrometry data suggest that no substances other than ginsenosides or eleutherosides were extracted from the plant material. Conclusion: Our data suggest that US ginseng products are correctly labeled as to plant genus; however, variability in concentrations of marker compounds suggests that standardization may be necessary for quality assurance and that characterization of herbal products should be considered in the design and evaluation of studies on herbal products. Am J Clin Nutr 2001;73: KEY WORDS Ginseng, ginsenosides, eleutherosides, standardization, Panax, Eleutherococcus, quality assurance, dietary supplements INTRODUCTION Since the passage of the Dietary Supplement Health Education Act of 1994, sales of dietary supplements have more than doubled (1); the current growth rate of herbal products is 11%/y (2). However, because dietary supplements do not have the same labeling requirements as do pharmaceuticals, there are concerns regarding their purity and potency (3). Reports that some herbal products contain potentially harmful adulterants (4 6) or have widely varying amounts of ingredients (7, 8) have heightened these concerns. These doubts prompted us to analyze a sampling of commercial ginseng products. We chose ginsengs because they are among the most popular herbal medicines worldwide and because they can be prepared from a variety of plants and, thus, are subject to botanical misidentification. Historically, the term ginseng referred to products prepared from the Panax species. More recently, however, the term is often applied to any herbal product with purported adaptogenic or restorative properties (9, 10). Korean and Chinese ginseng are prepared from the dried roots of Panax ginseng C.A. Meyer and American, Sanchi (Sanqi, Tienchi, or Tienqi), Japanese, and Vietnamese ginsengs are prepared from Panax quinquefolius L., Panax notoginseng (Burkill) F.H. Chen, Panax japonicus C.A. Meyer, and Panax vietnamensis Ha et Grushv., respectively. Each Panax species may have slightly different uses (9, 11) but all contain ginsenosides (or panaxosides) steroidal saponins that contain the 4 trans-ring rigid steroid skeleton and differ mainly by the number, type, and location of their sugar moieties (11, 12). Ginsenosides are unique to Panax species, are associated with the pharmacologic activity of Panax species, and are used as marker compounds for quality control (9, 11 14). The relative amounts of ginsenosides may also be used to differentiate between Panax species. For example, American ginseng has little or no ginsenoside Rf, has a lower ratio of ginsenoside Rg 1 to Rb 1 than Asian ginseng (15, 16), and, consistent with the pharmacology of ginsenosides Rg 1 (a weak stimulant of the central nervous system) and Rb 1 (a depressant of the central nervous system) 1 From the Department of Medical Pharmacology and Toxicology, the Division of Clinical Immunology, the Department of Nutrition, and the Division of Endocrinology and Clinical Nutrition, School of Medicine, University of California at Davis. 2 Supported by National Institutes of Health grant AI Address reprint requests to MR Harkey, Department of Medical Pharmacology and Toxicology, School of Medicine, University of California at Davis, Davis, CA martha@mother.com. Received May 16, Accepted for publication October 20, Am J Clin Nutr 2001;73: Printed in USA American Society for Clinical Nutrition 1101

2 1102 HARKEY ET AL (11), is considered to be more yin (ie, more balanced and less stimulating) than Asian ginseng (10). Siberian ginseng, prepared from the dried roots of Eleutherococcus (or Acanthopanax) senticosus Rupr. et maxim., is from the same family (Araliaceae) as but a different genus than the Panax species. Constituents of E. senticosus include 7 eleutherosides, which are chemically distinct from ginsenosides (17). Eleutherosides B (syringin) and E [( )-syringaresinol di-o- -Dglucoside] are 2 of the more abundant eleutherosides and are used as marker compounds for products of E. senticosus (18). In the present study, HPLC and HPLC-tandem mass spectrometry (LC-MS/MS) were used to identify and quantitate 7 ginsenosides (16, 19, 20) and 2 eleutherosides (18, 21) in each of the products. Concentrations and ratios of the respective marker compounds were then compared with information stated on the product label. MATERIALS AND METHODS Chemicals and reagents Twenty-five commercial products labeled as ginseng were purchased from a local health food store. Eight of the products were identified as containing P. ginseng, 4 as containing P. quinquefolius, 1 as containing P. notoginseng, 9 as containing E. senticosus, and 3 as containing mixtures of various ginsengs. Each product tested was unique; multiple samples of the same product or different lots were not tested. Ginsenoside standards (Rb 1, Rb 2, Rc, Rd, Re, Rf, and Rg 1 ) were obtained from Indofine Chemical Company, Inc (Somerville, NJ), and eleutheroside standards (B and E) from Extracts Plus (Carlsbad, CA). The identity and purity of the analytic standards were verified by LC- MS/MS. Solid-phase extraction columns (BondElut C-18, HF) were obtained from Varian Sample Preparation (Harbor City, CA) and high-purity solvents (HPLC grade acetonitrile, water, methanol, and trifluoroacetic acid) were obtained from Fisher Scientific (Pittsburgh). Standards and controls Stock solutions containing ginsenosides Rb 1,Rb 2, Rc, Rd, Re, Rf, and Rg 1 and eleutherosides B and E (each at a concentration of 1 g/l in methanol) were prepared and stored at 15 C. Inhouse stability studies showed these solutions to be stable for 4 mo. Calibration standards, quality-control samples, and fortified samples were prepared fresh daily from the stock solutions. Daily calibration plots were obtained by extracting ginsenoside or eleutheroside standards diluted with 1 ml 30% methanol in water to a final concentration of mg/l. Samples with concentrations above this range were diluted and reanalyzed. Both positive and negative quality-control samples were extracted and analyzed with each batch of commercial samples. For powdered samples, all the capsules in the bottle were opened and the contents mixed thoroughly before taking an aliquot for analysis to minimize any potential capsule-to-capsule variability. Sample preparation Ginsenosides were extracted from samples, standards, and controls by a modification of the method reported by Li et al (16). Two milliliters of 30% methanol in water was added to a 50-mg powdered sample or to a 0.1-mL liquid sample; the mixture was mixed by vortex for 10 s, heated at 50 C for 30 min, mixed by vortex again for 10 s, and then centrifuged (900 g, 15 min, room temperature). To determine extraction recovery, fortified samples were prepared by adding 10 or 100 g of each ginsenoside to a 50-mg sample of powdered P. ginseng, mixing by vortex for 10 s, adding 2 ml 30% methanol in water, and then following the procedure described above for extracting commercial samples. Recovery was determined by comparing the amount recovered from the fortified sample (ie, the amount in fortified samples less the amount in nonfortified samples) with unextracted solutions prepared at the fortified concentrations. Standards and controls were extracted with each batch of samples and were prepared by adding aliquots of standard solutions to a test tube containing 2 ml 30% methanol and processing as described above for the commercial samples. After centrifugation, the supernatant fluid was added to bonded-phase extraction columns (BondElut C-18, HF; Varian Sample Preparation) that had been preconditioned with 2 ml methanol followed by 2 ml water. The columns were washed with 2 ml 30% methanol; the ginsenosides were eluted in l ml methanol and then analyzed by HPLC. To ensure correct identification of ginsenosides, aliquots were also analyzed by LC- MS/MS after preparation of a 1:10 dilution of the extracts to approximate the initial mobile-phase composition (20). Eleutherosides were liquid extracted by using a modification of the procedure reported by Yat et al (18). Five milliliters of 80% methanol in water was added to a 100-mg powdered sample or to a 0.2-mL liquid sample. This mixture was mixed by vortex for 10 s and heated at 60 C for 30 min, and then the supernatant fluid was transferred to a separate test tube. The extraction was repeated with 5 ml 80% methanol at 60 C for 30 min and then the supernatant fluids were combined, evaporated, and reconstituted in 2 ml 0.05% trifluoroacetic acid in acetonitrile (1:4) and filtered through a 0.2- m nylon filter. Standards and controls were extracted in the same manner by adding aliquots of standard solutions to a test tube containing 5 ml 80% methanol. Extraction recovery was determined by fortifying a 100-mg powdered sample with 10 or 100 g of each eleutheroside and extracting as described above for the commercial samples. Identification and quantitation of ginsenosides and eleutherosides The identities of the ginsenosides and eleutherosides in the analytic standards and in the ginseng products were confirmed by negative-ion electrospray LC-MS/MS with a Finnigan LCQ Quadruple/Ion Trap Mass Spectrometer coupled to a TSP TP4000 HPLC (ThermoFinnigan Corp, San Jose, CA). The analytic column was a Zorbax C-18 (5 m, mm; Hewlett- Packard, Palo Alto, CA) and the mobile phase was varying concentrations of acetonitrile in water programmed by a system controller. Because of the complexity of the plants and because >30 ginsenosides have been identified in various preparations, the chromatographic method was optimized to eliminate these potential interferences (20). Ginsenosides and eleutherosides were quantitated by HPLC according to procedures reported for ginsenosides (16, 19) and eleutherosides (18, 21) with use of a liquid chromatograph system (model LC-M8A; Shimadzu, Columbia, MD) with a photodiode array detector (model SPD M10A; Shimadzu), controlled-temperature chamber (model CTO-10AS; Shimadzu), and autosampler (model ISS-100; Perkin-Elmer, Norwalk, CT). The analytic column was an Alltima C-18 (5 m, mm; Alltech Associates, Deerfield, IL) and the mobile phase was

3 VARIABILITY IN COMMERCIAL GINSENG PRODUCTS 1103 TABLE 1 Concentrations of ginsenosides in commercial ginseng products labeled as containing Panax species 1 varying concentrations of acetonitrile in water programmed with a Shimadzu model SCL 10A system controller. Ginsenosides were separated and quantitated by using a flow rate of 1.3 ml/min and a mobile phase of 21% acetonitrile for 20 min, 21 42% acetonitrile for min, 99% acetonitrile for 8 min, and reequilibration with 21% acetonitrile for 5 min. Quantitation was based on ultraviolet absorption at 203 nm. Eleutherosides were analyzed by using a flow rate of 1 ml/min and a mobile phase with a linear gradient of 10 30% acetonitrile for 0 15 min, 99% acetonitrile for 5 min, and reequilibration with 10% acetonitrile for 5 min. Quantitation was based on ultraviolet absorption at 220 nm. All analyses were performed at 30 C. Concentrations of ginsenosides and eleutherosides in commercial samples were determined by peak area with use of external standard analysis with a 5-point calibration curve extracted with each batch of samples. Positive and negative quality-control samples were added to each batch of samples. Ginsenoside samples were extracted and analyzed in duplicate on 2 separate occasions and eleutheroside samples were extracted and analyzed in duplicate on one occasion. Ginsenoside concentration Sample Labeled as Rb 1 Rb 2 Rc Rd Re Rf Rg 1 Rg 1 :Rb 1 Total Labeled amount 2 % % Powdered 3 C1 P. ginseng (65.7) C2 P. ginseng C3 P. notoginseng C4 P. ginseng C5 P. ginseng (136.8) C6 P. ginseng C7 P. ginseng (30.6) C8 P. ginseng and P. notoginseng C9 P. quinquefolius ND C10 P. ginseng, (31.7) P. quinquefolius, and E. senticosus C11 P. quinquefolius ND (57.5) Liquid 4 L1 P. ginseng (68.4) L2 P. quinquefolius ND L3 P. quinquefolius ND L4 P. ginseng L5 P. ginseng and (18.0) E. senticosus 1 Values are the mean of quadruplicate determinations. The mean CV for each determination was 9.2% (range: 1 20%). ND, not detected. 2 Percentage of labeled amount in parentheses. 3 Per 100 g product. 4 Per 100 ml product. RESULTS The purity of the ginsenoside and eleutheroside standards was verified to be >99% by LC-MS/MS. Concentrations of total ginsenosides (Rb 1,Rb 2, Rc, Rd, Re, Rf, and Rg 1 ) and eleutherosides (B and E) in the commercial products tested are shown in Tables 1 and 2, respectively. All of the commercial ginseng products contained the appropriate marker compounds and were correctly labeled as to genus of the plant material. Products labeled as containing Panax species contained ginsenosides, products labeled as containing E. senticosus contained eleutherosides, and products labeled as a mixture of Panax species and E. senticosus contained both ginsenosides and eleutherosides. Although it would be difficult to identify the species of Panax used in each preparation, the 4 products labeled as American ginseng had ginsenoside profiles typical of P. quinquefolius, namely no ginsenoside Rf and a low ratio of Rg 1 to Rb 1. There was, however, significant product-to-product variability in the amount of ginsenosides or eleutherosides present. Total ginsenoside concentrations varied 15-fold ( % by wt) in the Panax powders and capsules and 36-fold ( g/l) in the liquid extracts. Total eleutheroside concentrations varied 43- fold ( % by wt) in the Eleutherococcus powders and >200-fold ( g/l) in the liquid extracts. The mean CV for quadruplicate analysis of samples containing ginsenosides was 9.2% (range: 1 20%). The mean CV for the duplicate analysis of samples containing eleutherosides was 6.6% (range: 0 12%). Of the 25 samples tested, 11 were labeled as containing a specific concentration of ginsenosides or eleutherosides. Of these, 5 contained more than the labeled concentration of ginsenosides or eleutherosides and 6 contained less than the specified concentration. The concentrations found ranged from 10.8% to 327.7% of the labeled concentration. As shown in Table 3, several studies reported significant variability in the concentrations of ginsenosides and eleutherosides in commercial products. Because different analytic methods were used in the various studies, the values can be compared only gen-

4 1104 HARKEY ET AL TABLE 2 Concentrations of eleutherosides in commercial ginseng products labeled as containing Eleutherococcus senticosus 1 erally. The amounts of ginsenosides determined in the present study were within the same ranges shown in Table 3, but were somewhat lower than those reported for products in the United States (8) and Europe (22, 23) and for dried, intact ginseng roots sold for use in traditional Chinese medicines (13). These differences in ginsenoside concentrations are not surprising considering the variability in species used, the lack of standardized analytic methods, and the practice in Europe of fortifying herbal products with plant extracts to increase the concentration of marker compounds. The eleutheroside concentrations found in this study were also within the same range reported by others (17, 18). DISCUSSION Our analyses showed that the commercial ginseng products tested were appropriately labeled as to plant genus, but that variability among the products was considerable. In contrast with the Eleutheroside concentration Sample Labeled as B E Total Labeled amount 2 % % Powdered 3 C10 E. senticosus, P. ginseng, (11.9) and P. quinquefolius C12 E. senticosus (220.8) C13 E. senticosus C14 E. senticosus (327.7) C15 E. senticosus Liquid 4 L5 E. senticosus and P. ginseng (12.0) L6 E. senticosus L7 E. senticosus (244.3) L8 E. senticosus L9 E. senticosus (275.7) L10 E. senticosus Values are the mean of duplicate determinations. The mean CV for each determination was 6.6% (range: 0 12%). 2 Percentage of labeled amount in parentheses. 3 Per 100 g product. 4 Per 100 ml product. TABLE 3 Reported concentrations of ginsenosides and eleutherosides in commercial products 1 results of some studies (7, 8), all of the products we tested contained at least some amount of the appropriate marker compounds, either ginsenosides or eleutherosides. Furthermore, the marker compound profile for products labeled as American ginseng (ie, no ginsenoside Rf and a low ratio of Rg 1 to Rb 1 ) indicated that the correct Panax species (P. quinquefolius) was used to prepare the products. However, like other investigators, we found significant variability in concentrations of the marker compound, ie, measured concentrations were markedly different from the labeled concentrations. In general, Siberian ginseng products varied more than did Asian ginseng products and liquid extracts varied more than did powdered products. Concentrations of marker compounds in the products tested were lower than the labeled concentrations for 50% of the samples. This may not be surprising because it has been suggested that the increased demand for medicinal plants will lead to the harvesting of immature plants and thus a gradual lowering of ginsenoside concentrations, which may affect the overall quality of Total ginsenosides Total eleutherosides Sample and reference Powdered 2 Liquid 3 Powdered 2 Liquid 3 % % US ginseng products (present study) ( ) ( ) ( ) ( ) US ginseng products (8) 3.2 ( ) Swiss ginseng products (21) ( ) Swedish ginseng products (22) 6.18 ( ) Dried, intact roots of Panax ginseng, 4 ( ) Panax quinqefolius, and Panax notoginseng (13) US Siberian ginseng products (18) ( ) ( ) Dried, intact roots of Eleutherococcus senticosus (17) NA ( ) 1 x ; range in parentheses. Different analytic methods were used in the various studies; therefore, the values shown are only generally comparable. NA, not available. 2 Per 100 g product. 3 Per 100 ml product. 4 Only one liquid product was tested.

5 VARIABILITY IN COMMERCIAL GINSENG PRODUCTS 1105 the commercial products (24). Six of the products tested (samples C2, C8, L1, L2, L4, and L5) had total ginsenoside concentrations <1% (reported as g per 100 g or 100 ml product), which is low even if immature ginseng plants were used. P. ginseng contains 1% total ginsenosides after 1 y of growth and is typically not harvested until after 5 y of growth (25). There are relatively few published data on eleutheroside concentrations in either herbal products or intact E. senticosus plants. Although the E. senticosus products mentioned in the present study varied more than did the Panax products, the range of eleutheroside concentrations generally agreed with the values given in one report on Siberian ginseng products available in North America (18) and with the values reported in dried roots and stems of dried E. senticosus plants (17). The variability in concentration of the marker compound observed by all investigators suggests a need for standardization. European manufacturers have offered standardized herbal extracts for some time, at least to the extent that the extraction procedure is adjusted to ensure that a predetermined amount of a known constituent is present in each product. Similarly, many US manufacturers of herbal products now include marker compound concentrations on labels even though this is not currently required. However, the presence of specific concentrations of marker compounds (ie, chemically based standardization) may not guarantee pharmacologic activity. First, for most herbal products, data are insufficient to assign activity to a particular marker compound; therefore, there is no evidence on which to establish a threshold concentration. For example, products containing St John s wort have for some time been standardized to contain 0.04% hypericin; however, it is now believed that this constituent is not responsible for the antidepressant activity of St John s wort (26, 27). In the various Panax species, >30 ginsenosides have been identified, as have numerous other bioactive components including volatile oils ( 0.05%), 3 antioxidants (including maltol, salicylic acid, and vanillic acid), peptides, polysaccharides, polyacetylenic alcohols (eg, panaxynol), fatty acids, vitamins (eg, vitamin C, thiamine, riboflavin, vitamin B-12, and nicotinic acid), and minerals (eg, manganese, copper, cobalt, and arsenic) (9). To suggest that ginseng s biological activity is determined by only a few of its components may be too simplistic. In Asia, the quality of ginseng is not determined by the ginsenoside content but rather by the origin and age of the plant and the physical characteristics of the root. In all Panax species, ginsenoside concentrations are considerably higher in the flowers and leaves than in the root (9, 11, 16), but these portions are infrequently used. Similarly, ginsenoside concentrations are typically higher in the species P. notoginseng and P. japonicus than in the more highly valued P. ginseng (9, 13). This suggests that commercial value is not necessarily related to ginsenoside concentration. When standardization is achieved by fortifying with concentrated extracts, the desired activity of the final product may be altered or chemical residues from the extraction process may be present. In Germany, for example, P. ginseng products must be standardized to contain 1.5% ginsenoside Rg 1 (28); however, this concentration of the marker compound is not likely to be found in the natural dried root (13) and can thus be achieved only by fortifying natural root materials with extracts containing higher ginsenoside concentrations. 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